PROJECT SUMMARY The goal of this proposal is to understand the spatial and functional organization of the presynaptic periactive zone (PAZ), which is found adjacent to sites of synaptic vesicle release. The PAZ is a micron-scale structure, occupied by dozens of proteins that work together in multivalent assemblies to couple membrane remodeling to force-generating actin polymerization. Studies of PAZ proteins in many systems have suggested that these proteins act at multiple steps of the synaptic vesicle cycle as well as in other synaptic membrane functions (e.g. synaptic morphogenesis and receptor traffic). It remains unknown how the micron-scale organization and regulation of PAZ proteins direct their membrane and cytoskeleton remodeling activities to these different neuron-specific functions. We will use the Drosophila larval neuromuscular junction (NMJ), a powerful model synapse, to decipher how PAZ protein assemblies, activities, and cellular functions are linked. Using high- resolution imaging, we recently found that PAZ proteins occupy both overlapping and distinct domains within the PAZ, and that proper segregation of PAZ proteins between these domains depends on their multivalent interactions with each other. We have also recently described PAZ-dependent dynamic actin filament structures, which represent a direct readout of PAZ protein activities in these different domains. Using these tools, we will ask how synapses regulate PAZ protein activities and interactions in space and time, and how PAZ organization underlies its diverse neuron-specific functions, in response to synaptic activity and transmission. In Aim 1, we will determine how PAZ proteins are organized at resting and active synapses using complementary fixed, live, and super-resolution imaging methods, and develop new quantitative methods to describe their geometric relationships in PAZ domains. In Aim 2, we will test the hypothesis that synaptic actin patches represent clathrin-dependent synaptic vesicle recycling events, and identify the determinants of synaptic actin patch assembly and dynamics. In Aim 3, we will ask how PAZ organization and synaptic activity control diverse cell biological PAZ functions, including release site clearance and organization of cell adhesion complexes. Overall, our experiments will explain how organization of the PAZ into distinct subdomains underlies multiple functional and structural properties of synapses. PAZ proteins are implicated in multiple neurological disorders, so deciphering their in vivo functions will be critical for understanding the etiology of these human diseases.